A family of short RNA species ranging from in size 20 to 30 nucleotides has been identified and designated as small RNAs. Small RNAs are gene products and derived from double-stranded RNA (dsRNA) precursors that are processed by the ribonuclease type III enzyme Dicer. An increasing body of evidence has shown that these small noncoding RNAs play important roles in the regulation of gene expression including mRNA degradation, transcriptional repression, and chromatin modification. Science named this discovery as 2002's Breakthrough of the Year! No effort has been made to clone small RNAs expressed during mouse testicular development and spermatogenesis so far. Our hypothesis is that the male germ cells express numerous small RNAs in a spatial and temporal manner; many of these small RNAs are testes-specific and function as translational regulators and chromatin modifiers by regulating their target gene expression, or direct interaction, with heterochromatin during spermatogenesis. As an initial effort to test our hypothesis, we propose to perform the following studies: (1) cloning and sequencing of small RNAs expressed during testicular development and spermatogenesis; (2) determination of spatiotemporal expression profiles of the testes-specific small RNAs; (3) establishing of a Web-based testes-expressed small RNA database. The discovery of small RNAs expressed during development and involved in gene regulation opens a brand new avenue toward our better understanding of the mechanisms of gene regulation. For decades, reproductive biologists have been trying to dissect the regulatory mechanisms involved in gene expression regulation during spermatogenesis. The proposed work will identify most, if not all, small RNA species expressed during germ cell development, thus providing important preliminary data for the entire research community to further dissect molecular mechanisms that govern gene expression during spermatogenesis.